1. Field of the Invention
The present invention relates to recombinant aprotinin variants; processes to prepare the described peptide variants as homogeneously processed secretion products with transformed yeasts, and medicaments containing the recombinant aprotinin variants.
2. Background Information
Aprotinin is a well-investigated protein which contains 58 amino acids and acts to inhibit trypsin, chymotrypsin, plasmin and plasma kallikrein (H. Fritz and G. Wunderer, Drug. Res. 33, 479-494, 1983; W. Gebhard, H. Tschesche and H. Fritz, Proteinase Inhibitors, Barrett and Salvesen (eds.), Elseview Science Publ. BV 374-387, 1986).
Aprotinin obtained from bovine organs is the active substance in the medicament Trasylol.RTM. which is used for the treatment of various disorders such as, for example, hyperfibrinolytic hemorrhages and traumat.-hemorrhagic shock.
In addition, there are new clinical findings which show that the blood loss caused by fibrinolysis and coagulation in open-heart surgery can be considerably reduced by use of Trasylol.RTM. (W. van Oeveren et al. Ann. Thorac. Surg. 44, 640-634, 1987; D. Royston et al. Lancet II, 1289-1291, 1987; B. P. Bistrup et al., Lancet I, 366-367, 1988).
It has been possible to show that semisynthetically generated homologues of aprotinin which contain other amino acids in place of lysine in position 15 of the amino acid sequence have a profile of action and specificity of action which differ distinctly from those of aprotinin (Tschesche et al., U.S. Pat. No. 4,595,674; H. R. Wenzel et al. in Chemistry of Peptides and Proteins, Volume 3, 1985).
Some of these semisynthetic aprotinin homologues have, for example, a strongly inhibiting action on elastase from pancreas and leucocytes. Owing to this novel specificity of action, these aprotinin homologues can be used therapeutically for disorders caused by an increased release of elastase, such as, for example, development of emphysema, ARDS (adult respiratory distress syndrome) and rheumatoid arthritis.
Other aprotinin homologues with arginine in position 15 are characterized by an inhibitory action which is distinctly greater than that of aprotinin on plasma kallikrein, which is generally involved in the blood coagulation cascade.
Experience has shown that the yields achievable by semisynthetic modification of bovine aprotinin are small. It was therefore desirable to prepare by fermentation recombinant (rec.) gene products in prokaryotic microorganisms for the preparation of large amounts of aprotinin homologues advantageously using synthetically prepared genes (Auerswald et al., Patent EP 01238993 A2; B.v. Wilcken-Bergmann et al., EMBO J. 5, 3219-3225, 1986).
For example, the expression systems used for the preparation of rec. aprotinin variants in E. coli K 12 strains were ones which accumulate, in the form of intracellular inclusion bodies, the aprotinin mutein as a fusion protein, which is formed within the cell, with a suitable fusion partner such as, for example, the N-terminal peptide portion of MS 2 replicase (E. A. Auerswald et al., Biol. Chem. Hoppe Seyler 369, 27-35, 1988).
Besides these, it is also possible to use E. coli expression/secretion systems which make possible, by fusion of the aprotinin mutein with suitable gene sequences for secretory signal peptides, such as, for example, the OmpA signal sequence or the phoA signal sequence, the secretion of inhibitory aprotinin variants into the periplasm of the bacterial cell (personal communication, Dr. W. Bruns--Bayer AG; C. G. Marks et al., J. Biol Chem. 261, 7115-7118, 1986).
Among eukaryotic systems, yeast expression systems are particularly suitable for the genetically engineered preparation of rec. aprotinin variants, in which the gene product is either accumulated within the cell or, as fusion with a suitable secretory signal sequence from yeasts, passed through the secretion pathway and, after cleavage off by a membrane protease, exported as inhibitory substance into the culture medium. Examples of suitable signal sequences which can be used for the secretion are the signal sequences of alpha-mating factor, of alpha-amylase, of glucoamylase or of invertase.
However, besides E. coli and yeasts, it is also possible to use many other prokaryotic and eukaryotic expression/secretion systems for the preparation of rec. aprotinin variants, such as, for example, Bacilli, Staphylococci or Aspergilli.
As the abovementioned examples show, the expression of aprotinin muteins in various prokaryotic and eukaryotic systems is the state of the art.
It is advantageous in this connection, with a view to preparation on the industrial scale, for the aprotinin variants to be obtained not in a form accumulated within a cell but, by utilization of the secretion mechanisms intrinsic to the system, as active substances exported into the fermentation medium.
When yeast systems are used, for this purpose signal sequences of secretory yeast proteins, such as, for example, of alpha-amylase, of glucoamylase, of invertase or of alpha-mating factor, are fused by genetic engineering to the N terminus of the aprotinin variants.
The enzymatic cleavage of the signal peptide of the N-terminus of the aprotinin variants takes place on membrane transfer by an enzyme which is intrinsic to yeast and which recognizes a specific cleavage sequence (at the C terminus of the signal peptide) (see Review Article R. C. Das and J. L. Schultz, Biotechn. Progress 3, 43-48, 1987).
However, it has emerged in the case of the rec. aprotinin variants with the natural N-terminal amino acid sequence "Arg-Pro-Asp" which are expressed in yeasts that, owing to incorrect cleavage off of the abovementioned signal peptides, the secreted material has variable N-terminal additions of amino acid of the various fused signal peptides. A uniformly and correctly processed secretion product suitable for purification is not found.
The partial or complete faulty processing of secretion products is also described in the literature for other heterologous proteins expressed as fusion products with secretory signal sequences intrinsic to yeasts (R. C. Das and J. L. Schultz, Biotechn. Progress 3, 43-48, 1987; P. J. Barr et al., J. Biol. Chem. 263, 16471-16478, 1988).